www.nature.com/cr www.cell-research.com ARTICLE OPEN NDUFAB1 confers cardio-protection by enhancing mitochondrial bioenergetics through coordination of respiratory complex and supercomplex assembly Tingting Hou1, Rufeng Zhang1, Chongshu Jian1, Wanqiu Ding1, Yanru Wang1, Shukuan Ling2,QiMa1, Xinli Hu1, Heping Cheng1 and Xianhua Wang1 The impairment of mitochondrial bioenergetics, often coupled with exaggerated reactive oxygen species (ROS) production, is a fundamental disease mechanism in organs with a high demand for energy, including the heart. Building a more robust and safer cellular powerhouse holds the promise for protecting these organs in stressful conditions. Here, we demonstrate that NADH: ubiquinone oxidoreductase subunit AB1 (NDUFAB1), also known as mitochondrial acyl carrier protein, acts as a powerful cardio- protector by conferring greater capacity and efficiency of mitochondrial energy metabolism. In particular, NDUFAB1 not only serves as a complex I subunit, but also coordinates the assembly of respiratory complexes I, II, and III, and supercomplexes, through regulating iron-sulfur biosynthesis and complex I subunit stability. Cardiac-specific deletion of Ndufab1 in mice caused defective bioenergetics and elevated ROS levels, leading to progressive dilated cardiomyopathy and eventual heart failure and sudden death. Overexpression of Ndufab1 effectively enhanced mitochondrial bioenergetics while limiting ROS production and protected the heart against ischemia-reperfusion injury. Together, our findings identify that NDUFAB1 is a crucial regulator of mitochondrial energy and ROS metabolism through coordinating the assembly of respiratory complexes and supercomplexes, and thus provide a potential therapeutic target for the prevention and treatment of heart failure. Cell Research (2019) 29:754–766; https://doi.org/10.1038/s41422-019-0208-x INTRODUCTION sites in the complexes from oxidative damage, and stabilize Being so-called “powerhouses”, mitochondria comprise up to 40% individual complexes.11,15–21 Interestingly, SC formation is dyna- of the heart mass and are central to cardiac bioenergetics.1,2 As mically regulated in accordance with changes in cellular hazardous by-products of energy metabolism, reactive oxygen metabolism,22–25 and deficiency of SCs is associated with heart species (ROS) are also emitted from the respiratory electron failure.26 Therefore, enhancing SC formation might afford an transport chain (ETC), and excessive ROS emission causes effective therapeutic strategy to make the mitochondria more oxidative damage to proteins, lipids, and even genetic materials.3,4 efficient and safer powerhouses. Studies have shown that >50% of individuals with mutations in NADH:ubiquinone oxidoreductase subunit AB1 (NDUFAB1), also genes encoding mitochondrial proteins eventually develop known as mitochondrial acyl carrier protein,27 plays a multi- cardiomyopathy,5 suggesting that defective cardiac bioenergetics functional role in the organelle. It not only participates in the as well as its interlinked ROS metabolism is a fundamental disease synthesis of lipoic acid in the type II fatty acid biosynthetic mechanism in the heart.6,7 As such, building a more robust cellular pathway (FAS II),28,29 but also acts as an accessory subunit of powerhouse with decreased ROS emission holds the promise for complex I with a 2:1 stoichiometry.30 A recent study has shown protecting the heart in stressful conditions. that NDUFAB1 interacts with seven mitochondrial LYR motif- The most important player in mitochondrial energy production containing (LYRM) proteins: LYRM1, LYRM2, LYRM4/ISD11, LYRM5, is the ETC, which consists of four multi-heteromeric complexes LYRM7/MZM1L, SDHAF3/ACN9, and FMC1/C7orf55.31 Most of (complexes I–IV) in the inner membrane of the organelle. They them are linked to various mitochondrial metabolic processes, catalyze outward proton movement to build a transmembrane such as the assembly of complex II (for SDHAF3/ACN9) and electrochemical gradient that drives ATP synthase (complex V) for complex V (for FMC1/C7orf55), deflavination of the electron- ATP synthesis.8 Individual ETC complexes can also organize into transferring flavoprotein (for LYRM5), and biosynthesis of iron- supercomplexes (SCs) of different composition and stoichiometry, sulfur (FeS) centers (for LYRM4/ISD11).31 In yeast cells which lack which have recently been visualized by cryo-electron micro- complex I, Van et al. have shown that NDUFAB1 interacts with and scopy.9–14 Such SCs are thought to channel electron transfer stabilizes the FeS biogenesis complex to regulate the assembly of more efficiently, limit ROS production, protect vulnerable ETC complexes II and III and SCs.32 In addition, previous evidence in 1State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Institute of Molecular Medicine, Peking University, Beijing 100871, China and 2State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, Beijing 100094, China Correspondence: Heping Cheng ([email protected]) or Xianhua Wang ([email protected]) Received: 16 January 2019 Accepted: 2 July 2019 Published online: 31 July 2019 © The Author(s) 2019 Article 755 293T cells has also suggested a role of NDUFAB1 in complex I and the inter-ventricular septum was decreased whereas the left assembly and the existence of a complex I-independent mechan- ventricular diameter and volume were increased with age in cKO ism for Ndufab1 knockout-induced cell death.33 Thus, NDUFAB1 hearts (Supplementary information, Fig. S2b, c). These findings might be a core player in orchestrating mitochondrial energy indicate that cardiac-specific ablation of NDUFAB1 leads to dilated biogenesis. cardiomyopathy accompanied by cardiomyocyte hypertrophy, In this study, we aimed to test the potential protective role of interstitial fibrosis, and systolic and diastolic dysfunction, even- NDUFAB1 in the mammalian heart using cardiac-specific Ndufab1 tually resulting in heart failure and sudden death, revealing an knockout (cKO) and transgenic overexpression (TG) mouse essential role of NDUFAB1 in cardiac function. models. We find that NDUFAB1 coordinates the assembly of individual ETC complexes and SCs and acts as a crucial Mitochondrial dysfunctions in Ndufab1 cKO heart endogenous regulator of the efficiency and capacity of mitochon- To investigate the mechanism underlying the cardiomyopathy drial energy metabolism as well as ROS emission. More and heart failure induced by NDUFAB1 ablation, we next assessed importantly, NDUFAB1 exerts a cardio-protective effect when the changes of cardiac mitochondrial functions at different ages of heart is subjected to ischemia-reperfusion (IR) injury. Our findings cKO mice. Electron microscopic analysis revealed that cardiac substantiate that targeting mitochondria for greater bioenergetic mitochondria were in disarray along the sarcomeres with some capacity and repressed ROS emission with enhanced SC formation exhibiting irregularities in the cristae formation in 16-week-old is an effective strategy for cardio-protection, and mark NDUFAB1 cKO mice, whereas mitochondrial morphology was apparently as a potential therapeutic target for the prevention and treatment normal at 6 weeks, when the cardiac function hadn’t been of cardiomyopathy. impaired yet (Fig. 2a). The mitochondrial DNA content and cross- sectional area of individual mitochondria didn’t display detectable changes whereas the total mitochondrial volume fraction and RESULTS mitochondrial number were slightly increased in 16-week-old cKO Cardiac-specific ablation of NDUFAB1 causes progressive dilated mice (Supplementary information, Fig. S3). Measuring mitochon- cardiomyopathy leading to heart failure drial membrane potential (ΔΨm) in isolated cardiomyocytes with NDUFAB1 was widely expressed in different tissues and was the potential-sensitive fluorescent probe tetramethyl rhodamine particularly enriched in the heart (Supplementary information, methyl ester (TMRM) showed that NDUFAB1 ablation significantly Fig. S1a). Whole-body knockout of Ndufab1 was embryonic lethal decreased ΔΨm even at 6 weeks (Fig. 2b) when the heart 1234567890();,: (Supplementary information, Fig. S1b, c), showing that NDUFAB1 morphology and function were normal (Fig. 1), indicating is essential for developmental viability. To explore the potential mitochondrial dysfunction is an early event in cKO mice preceding cardiac functions of NDUFAB1, we generated a cardiac-specific the onset of cardiomyopathy. Measurements with the fluorescent knockout mouse model (cKO) wherein exon 3 of Ndufab1 was probe mitoSOX showed that mitochondrial ROS level was flanked by loxP sites (Ndufab1flox/flox, Supplementary information, significantly elevated in cKO cardiomyocytes compared to WT at Fig. S1b). The Ndufab1flox/flox mice were cross-bred with Mlc2v-Cre 10 weeks, and increased by 72% at 14–16 weeks, reaching a level mice to allow cardiomyocyte-specific deletion of Ndufab1,as comparable to that induced by 5 μg/mL antimycin A (Fig. 2c). The previously described.34 As shown in Fig. 1a and Supplementary cellular ATP level, as measured with the luciferin luminescence information, Fig. S1d, cardiac expression of Ndufab1 was assay, showed a trend of decrease in cKO mice from 6 to 8 weeks decreased by ~90% at the protein level in cKO cardiomyocytes old and was significantly lowered in 14–16-week or older cKO compared to wild-type (WT) cells. The